Zn^(2+)对PPR蛋白锌指结构域原核表达的影响

Effect of Zn^(2+) on Prokaryotic Expression of Zinc Finger Domain of PPR Protein

目的研究Zn^(2+)对PPR蛋白锌指结构域的原核表达、纯化过程的影响,并对Zn^(2+)的作用机制进行了初步探索。方法采用分子克隆的方法构建PPR蛋白锌指结构域及其突变体的His-SUMO融合蛋白表达质粒,研究Zn^(2+)对其原核表达及Ni-柱亲和纯化过程的影响,并利用快速诱导法对Zn^(2+)的作用机制进行了初步探索。结果培养基中添加1 mmol/L Zn^(2+)可明显提高融合蛋白的稳定性;亲和纯化时在上清中添加10 mmol/L EDTA可明显提高亲和纯化效率;Zn^(2+)作用机制的初探实验表明只有Zn^(2+)可作用于锌指结构位点,并帮助稳定锌指结构域。结论 Zn^(2+)可作用于PPR蛋白的锌指结构位点,并使锌指结构变得稳定;在蛋白原核表达时添加一定浓度的Zn^(2+)可明显提高蛋白稳定性。

Objective To study the effect of zinc ion on the prokaryotic expression and purification process of the zinc finger domain of PPR protein,and the action mechanism of zinc ions was explored.Methods The zinc finger domain of PPR protein and its mutant were cloned into His-SUMO fusion protein expression vectors by molecular cloning,and the effect of zinc ions on the prokaryotic expression and Ni-column affinity purification process were investigeted.The action mechanism of of zinc ions was explored by rapid induction method.Results The addition of 1 mmol/L of zinc ion in the medium can significantly improve the stability of the fusion protein.Adding 10 mmol/L of EDTA to the supernatant can significantly improve the affinity purification efficiency.The preliminary study on the action mechanism of zinc ions showed that only zinc ions can act on the zinc finger domain and help stabilize its structure.Conclusion Zinc ion can act on the zinc finger structure site of PPR protein and make its structure stable;adding a certain concentration of zinc ion in the process of prokaryotic expression can significantly improve the stability of the protein.